Dermatological compositions and methods

ABSTRACT

Disclosed are methods and compositions for regulating the melanin content of mammalian melanocytes; regulating pigmentation in mammalian skin, hair, wool or fur; treating or preventing various skin and proliferative disorders; by administration of various compounds, including alcohols, diols and/or triols and their analogues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of 09/085,917, filed May 28, 1998,now U.S. Pat. No. 6,623,724, which is a continuation-in-part ofPCT/US98/05346 filed Mar. 18, 1998, which is a continuation-in-part ofPCT/US97/16642 filed Sep. 18, 1997, which is a continuation-in-part ofapplication Ser. No. 08/933,143 filed Sep. 18, 1997 now abandoned, whichis a continuation-in-part of application Ser. No. 60/026,577 filed Sep.18, 1996, of application Ser. No. 60/035,947 filed Jan. 21, 1997, ofapplication Ser. No. 60/036,863 filed Feb. 4, 1997, and of applicationSer. No. 60/048,597 filed Jun. 4, 1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to regulating the melanin content ofmammalian melanocytes; regulating pigmentation in mammalian skin, hair,wool or fur; restoring pigmentation to grey hair; treating or preventingvarious skin and proliferative disorders; by administration of variouscompounds, including alcohols, diols and/or triols and their analogues.

2. Description of Related Art

U.S. Pat. No. 5,352,440 is directed to increasing melanin synthesis inmelanocytes and increasing pigmentation by administration of certaindiacylglycerol compounds.

U.S. Pat. No. 5,532,001 is directed to increasing pigmentation inmammalian skin via administration of certain DNA fragments.

U.S. Pat. No. 5,554,359 is directed to increasing levels of melanin inmelanocytes by administration of lysosomotropic agents.

SUMMARY OF THE INVENTION

The present invention provides a method for increasing the melanincontent of mammalian melanocytes, which comprises administering to saidmelanocytes an effective amount of a C₃-C₅₀ diol, which may be aliphaticor aromatic, linear, branched, mono-, bi- or polyclicic, saturated orunsaturated, unsubstituted, mono- or polysubstituted.

Another aspect of the present invention concerns a method for increasingor restoring pigmentation in mammalian skin, hair or wool, whichcomprises administering to said mammal an effective amount of one ormore compounds described above.

Another aspect of the present invention concerns a method for treating askin proliferative disorder or a disorder of keratinization in a mammal,which comprises administering to a mammal in need of such treatment aneffective amount of one or more compounds described above.

A further aspect of the present invention concerns a method forpreventing a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchpreventive treatment an effective amount of one or more compoundsdescribed above.

An additional aspect of the present invention concerns a method fortreating a tumorous or cancerous disorder whereby application of one ormore of the compounds described above results in reversal of saiddisorder by virtue of induction of differentiation of cancerous ortumorous cells to a less- or non-proliferative phenotype. Thesecancerous or tumorous disorders include, but are not limited to,proliferative disorders of a dermatological nature.

In another aspect, the present invention provides a composition forincreasing the melanin content of mammalian melanocytes, whichcomprises:

a) an effective amount of one or more compounds described above; and

b) a suitable carrier.

In another aspect, the present invention provides a composition fortreating a skin proliferative disorder or a disorder of keratinization,which comprises:

a) an effective amount of one or more compounds described above; and

b) a suitable carrier.

In yet another aspect, the present invention provides a composition forpreventing a skin proliferative disorder, which comprises:

a) an effective amount of one or more compounds described above; and

b) a suitable carrier.

The present invention additionally provides a method for increasing themelanin content of mammalian melanocytes, which comprises administeringto said melanocytes an effective amount of one or more compounds havingthe following structure:

wherein

each X is independently selected from a single or double bond; or agroup containing from one atom to twenty atoms, at least one of which iscarbon, nitrogen, oxygen or sulfur;

each R₁ is independently selected from hydrogen; halogen; an acyl oramino acyl group containing from one atom to twenty atoms, at least oneof which is carbon, nitrogen, oxygen, or sulfur; or a group containingfrom one atom to twenty atoms, one of which is carbon, nitrogen, oxygen,or sulfur;

R₂ is a linear, branched or unbranched, cyclic, bicyclic or polycyclicgroup containing from one atom to fifty atoms, at least one of which iscarbon, nitrogen, oxygen, or sulfur; and

each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃—, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25;

and pharmaceutically acceptable salts or prodrugs thereof, with theproviso that with reference to the first listed structure only, when theX to which R₁ is attached is a single bond and each R is acyl and one ofR₁ is hydroxymethyl (HOCH₂—), then the sum of carbon atoms in R₁ isgreater than one.

Another aspect of the present invention concerns a method for increasingor restoring pigmentation in mammalian skin, hair or wool, whichcomprises administering to said mammal an effective amount of one ormore compounds depicted above.

Another aspect of the present invention concerns a method for treating askin proliferative disorder or a disorder of keratinization in a mammal,which comprises administering to a mammal in need of such treatment aneffective amount of one or more compounds depicted above.

A further aspect of the present invention concerns a method forpreventing a skin proliferative disorder or a disorder of keratinizationin a mammal, which comprises administering to a mammal in need of suchpreventive treatment an effective amount of one or more compoundsdepicted above.

An additional aspect of the present invention concerns a method fortreating a tumorous or cancerous disorder whereby application of one ormore of the compounds depicted above results in reversal of saiddisorder by virtue of induction of differentiation of cancerous ortumorous cells to a less- or non-proliferative phenotype. Thesecancerous or tumorous disorders include, but are not limited to,proliferative disorders of a dermatological nature.

In another aspect, the present invention provides a composition forincreasing the melanin content of mammalian melanocytes, whichcomprises:

a) an effective amount of one or more compounds depicted above; and

b) a suitable carrier.

In another aspect, the present invention provides a composition fortreating a skin proliferative disorder or a disorder of keratinization,which comprises:

a) an effective amount of one or more compounds depicted above; and

b) a suitable carrier.

In yet another aspect, the present invention provides a composition forpreventing a skin proliferative disorder, which comprises:

a) an effective amount of one or more compounds depicted above; and

b) a suitable carrier.

In yet another aspect, the present invention provides a method ofaltering or restoring pigmentation in mammalian skin, hair, wool or fur,which comprises administering to a mammal an effective amount of acompound which alters cellular production of nitric oxide, wherein anincrease in nitric oxide production results in increased pigmentation,and a decrease in nitric oxide production results in decreasedpigmentation.

In yet another aspect, the present invention provides a method ofaltering pigmentation in mammalian skin, hair, wool or fur, whichcomprises administering to a mammal an effective amount of a compoundwhich alters cellular production of cyclic guanosine monophosphate,wherein an increase in cyclic guanosine monophosphate production resultsin increased pigmentation, and a decrease in cyclic guanosinemonophosphate production results in decreased pigmentation.

In yet another aspect, the present invention provides a method ofaltering pigmentation in mammalian skin, hair, wool or fur, whichcomprises administering to a mammal an effective amount of a compoundwhich alters cellular activity of protein kinase G, wherein an increasein protein kinase G activity results in increased pigmentation, and adecrease in protein kinase G activity results in decreased pigmentation.

In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular production of nitric oxide, wherein if such production isaltered, then the pigmentation in mammalian melanocytes is altered.

In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular production of cyclic guanosine monophosphate, wherein if suchproduction is altered, then the pigmentation in mammalian epidermalmelanocytes is altered.

In yet another aspect, the present invention provides a method ofidentifying a substance which alters pigmentation in mammalianmelanocytes, which comprises evaluating the effect the substance has oncellular activity of protein kinase G, wherein if such activity isaltered, then the pigmentation in mammalian epidermal melanocytes isaltered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are printouts from an Oncor Imaging Systems ofFontana-Masson stained guinea pig skin biopsy samples as described inExample 5.

FIG. 2 is a series of bar graphs depicting the structure activityresults obtained in Example 7.

FIGS. 3A-3D are printouts of normal human epidermal melanocytes andmelanosomes as described in Example 8.

FIG. 4 is a series of bar graphs depicting the structure activityresults obtained in Example 12.

FIGS. 5A-5B are photographs of treated guinea pig skin as described inExample 13.

FIGS. 6A-6D are printouts as described in Example 13.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unique observation that certaincompounds effectively and efficiently induce melanogenesis in mammaliancells, which has several consequences. First, increasing melanogenesisleads to increasing the melanin content of melanocytes, and henceresults in increased pigmentation or darkened color of the skin, hairwool or fur. Thus, the present invention is useful in the treatment ofhypopigmentation disorders, such as albinism, vitiligo, etc. It is alsobelieved that increasing the pigmentation of skin according to thepresent invention will protect such skin from subsequent UV lightdamage, sunburn, photoaging and development of skin cancers. Finally,since the methods and compositions described herein inducedifferentiation of a melanoma cell line, the present invention may beused to treat hyperproliferative disorders such as actinic keratosis,basal cell carcinoma, squamous cell carcinoma, fibrous histiocytoma,dermatofibrosarcoma protuberans, hemangioma, nevus flammeus, xanothoma,Kaposi's sarcoma, mastocytosis, mycosis fungoides, lentigo, nevocellularnevus, lentigo maligna, malignant melanoma, and metastatic carcinoma.

The present methods and compositions are also useful in the treatment ofdiseases characterized by inflammation and disturbance ofkeratinization, including psoriasis vulgaris, psoriasis eosinophilia,acne vulgaris, acne conglobata, acne fulminans, osteoma cutis,nodulocystic acne, cystic acne and benign and premalignant dermatoses.

The active compounds according to the present invention are the C₃-C₅₀diols described above (by “diol” is meant a compound which has at leasttwo, but permissibly more, —OH groups). Preferably, the active have oneof the six structures depicted above. More preferably, X isindependently selected from a single bond; or C₁-C₁₀ alkylene, C₂-C₁₀alkenylene, or C₂-C₁₀ alkynylene, each of which may contain one or moredifferent heteroatoms or heteroatoms of the same type. More preferablyeach R₁ is independently selected from hydrogen; fluoro; chloro; orC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₇-C₂₀ aralkyl, C₈-C₂₀aralkenyl, C₈-C₂₀ aralkinyl, or C₆-C₂₀ aryl, each of which may containone or more different heteroatoms or heteroatoms of the same type, orcarboxyl, carboxamido, carbalkoxy, sulfamido, sulfonamido; hydroxyl, oramino. More preferably R₂ contains from two to twenty carbon atoms, eachmay contain one or more different heteroatoms or heteroatoms of the sametype.

The preparation of the present compounds would be apparent to one ofordinary skill, and many of them are commercially available.Representative preferred compounds include, but are not limited to:

-   1,2-Ethanediol-   1,2-Propanediol(Propylene Glycol)-   (S)-(+)-1,2-Propanediol[(S)-(+)-1,2-Propylene Glycol]-   1,3-Propanediol-   2,3-Dimethyl-2,3-Butanediol-   2,3-Dimethyl-1,2-Butanediol-   1-Phenyl-1,2-Propanediol-   2-Methyl-1,3-Propanediol-   1,2-Butanediol-   1,3-Butanediol-   1,4-Butanediol-   2,3-Butanediol-   (2R,3R)-(−)-2,3-Butanediol-   (2S,3S)-(+)-2,3-Butanediol-   2,3-meso-Butanediol-   1,2-Pentanediol-   1,4-Pentanediol-   1,5-Pentanediol-   2,4-Pentanediol-   1,2-cis-cyclopentanediol-   1,2-trans-cyclopentanediol-   1,2-cis-cyclohexaneanediol-   1,2-trans-cyclohexanediol-   1,2-dihydroxy-4,5-cyclohexanediol carbonate-   1,2,4,5-tetrahydroxycyclohexane-   1,2-Hexanediol-   1,5-Hexanediol-   1,6-Hexanediol-   2,5-Hexanediol-   1,2-Heptanediol-   1,7-Heptanediol-   7-Octene-1,2-diol-   1,2-Octanediol-   1,8-Octanediol-   1,2-Nonanediol-   1,9-Nonanediol-   1,2-Decanediol-   1,10-Decanediol-   1,2-Dodecanediol-   1,12-Dodecanediol-   1,2-Tetradecanediol-   1,14-Tetradecanediol-   1,2-Hexadecanediol-   1,16-Hexadecanediol-   Glycerol-   1,2,4-Butanetriol-   1,2,3-Trihydroxyhexane-   1,2,6-Trihydroxyhexane-   1,2,3-Heptanetriol-   β-estradiol-   azabicyclo-(2,2,1)-heptanediol-3-one-   1,4-dioxane-2,3-diol-   5-norbornene-2,2-dimethanol-   norbornane-2,2-dimethanol-   2,3-norbornanediol (exo or endo or cis or trans)-   2,3-cis-exo-norbornanediol-   α-norborneol-   2-norbornanemethanol-   norbornane-   borneol-   camphor-   camphene-   camphane-   norbornane acetic acid-   norbornane-carboxylic acid-   norbornane-dicarboxylic acid-   2-endo-hexadecylamino-5-norbornene-2-exo-methanol-   2-endo-hexadecylamino-5-norbornene-2,3-exo-dimethanol-   2-(propyl-1,2-diol)-norbornane-   1,2-dithiane-trans-4,5-diol-   2,3-pyridinediol-   2,3-pyridinediol hydrogen chloride-   2,3-pyridinediol glycolic acid-   2,3-dipyridyl-2,3-butanediol-   2,2,4,4-tetramethyl-1,3-cyclobutanediol-   norborneol-   2,7-norbornanediol-   2,5,7-norbornanetriol-   2,6,7-norbornanetriol-   2-hydroxy-2-norbornanemethanol-   1-(exo-2-norbornyl-)-propan-1,2-diol-   1-(endo-2-norbornyl-)-propan-1,2-diol-   methyl-5-norbornene-2,3-dimethanol-   2-norbornaneacetic acid-   1,2-cis-cyclohexanedimethanol-   3-cyclohexane-1,1-dimethanol-   1,4-cyclohexanedimethanol-   pentaerylthritol-   pinane-   pinaneol-   2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and    [1S,2S,3R,5S]-[+]-pinanediol])-   (1R)-(−)-trans-pinane-1,10-diol-   (1S,2S,5S,)-2-hydroxy-3-pinanone-   (−)-isopinocampheol-   (S)-cis-verbenol-   bornane-   borneol-   2,3-cis/exo-bornanediol-   2,3-trans-bornanediol-   camphanediol-   camphenediol-   cis-p-menthane-3,8-diol-   trans-p-menthane-3,8-diol-   sobrerol(trans-p-meth-6-ene-2,8-diol)-   α-terpineol-   terpinen-4-ol-   (−)-cis-myrtanol[(1S,2R)-10-Pinanol]-   (+)-trans-myrtanol[(1R,2R)-10-Pinanol]-   (−)-trans-myrtanol[(1S,2S)-10-Pinanol]-   (−)-myrtenal[(1R)-2-Pinen-10-al]-   (−)-myrtenol[(1R)-2-Pinene-10-ol]-   carveol[p-mentha-6,8-dien-2-one]-   menthol

Particularly preferred compounds of this invention are2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]; 2,3-cis/exo-bornanediol;5-norbornene-2,2-dimethanol; norbornane-2,2-dimethanol;2-hydroxy-2-norbornanemethanol; 1-(exo-2-norbornyl-)-propan-1,2-diol;and 1-(endo-2-norbornyl-)-propan-1,2-diol. Other preferred compounds are(1S,2S,5S,)-2-hydroxy-3-pinanone; 2,3-trans-pinanediol;(1R)-(−)-trans-pinane-1,10-diol; 2,3-trans-bornanediol;cis-p-menthane-3,8-diol; trans-p-menthane-3,8-diol;1,2-cis-cyclopentanediol, 2,3-cis/exo-norbornanediol;2-norbornanemethanol; (1R)-(−)-myrtenol, and3,3-dimethyl-1,2-butanediol.

The methods and compositions of the present invention contemplate theuse of one or more of the above-mentioned compounds as an activeingredient for various uses. In a preferred embodiment, the activeingredient(s) is combined with an acceptable carrier to form a topicalformulation which may be placed on the skin for dermatological uses.Topical formulations may include ointments, lotions, pastes, creams,gels, drops, suppositories, sprays, liquids, shampoos, powders andtransdermal patches. Thickeners, diluents, emulsifiers, dispersing aidsor binders may be used as needed. Preferably, one function of thecarrier is to enhance skin penetration of the active ingredient(s), andshould be capable of delivering the active ingredient(s) to melanocytesunder in vivo conditions. Suitable carriers are well known to one ofordinary skill, and include liposomes, ethanol, dimethylsulfoxide(DMSO), petroleum jelly (petrolatum), mineral oil (liquid petrolatum),water, dimethylformamide, dekaoxyethylene-oleylether, oleic acid,2-pyrrolidone and Azone® brand penetration enhancer (Upjohn). Aparticularly preferred composition includes an active ingredient(s) asdescribed above, with one of 2-pyrrolidone, oleic acid and/or Azone® aspenetration enhancer, solubilized in a base of water, ethanol, propanoland/or propylene glycol (the latter component having properties of acarrier, penetration enhancer and an active ingredient as describedherein). Depending on the specific application, the compositions of thepresent invention may also include other active ingredients, as well asinert or inactive ingredients.

Particularly preferred formulations include an active ingredient(s) inconjunction with one or more melanogenesis-enhancing agents such asα-hydroxy acids, salts and derivatives thereof; α-keto acids, salts andderivatives thereof; β-hydroxy acids, salts and derivatives thereof;retinoids, salts and derivatives thereof; Vitamin A and relatedcompounds; acids; phenol; and methoxypropyl-gluconamide, as more fullydescribed in co-pending application Ser. No.09/055,274 filed Apr. 6,1998 entitled “Dermatological Formulations and Methods”, the contents ofwhich are incorporated herein by reference.

The dose regimen will depend on a number of factors which may readily bedetermined, such as severity and responsiveness of the condition to betreated, but will normally be one or more doses per day, with a courseof treatment lasting from several days to several months, or until acure is effected or a diminution of disease state is achieved, or acosmetically desired degree of melanogenesis (tanning) is achieved,depending on the application. One of ordinary skill may readilydetermine optimum dosages, dosing methodologies and repetition rates. Ingeneral, it is contemplated that topical formulations (such as creams,lotions, solutions, etc.) will have a concentration of active ingredientof from about 0.01% to about 50%, preferably from about 0.1% to about10%. In general, it is contemplated that unit dosage form compositionsaccording to the present invention will contain from about 0.01 mg toabout 100 mg of active ingredient, preferably about 0.1 mg to about 10mg of active ingredient.

Another aspect of the present invention is based on the observation thatthe subject compounds which stimulate melanin production act via theNitric Oxide/cyclic Guanosine monophosphate/Protein Kinase G(“NO/cGMP/PKG”) pathway. Thus, the present invention includes not onlythe compounds described above, but any compound which acts via theNO/cGMP/PKG pathway to stimulate melanin synthesis by increasingcellular production of NO, cGMP or PKG. Conversely, agents whichdecrease cellular production of NO, cGMP or PKG will decrease orsuppress melanin production and pigmentation in mammalian skin, hair,fur or wool, and the present invention is also directed to thosecompositions and methods. Such is useful in, for example, the lighteningof skin, hair, wool or fur for cosmetic purposes, or the treatment ofhyperpigmentation or uneven pigmentation disorders such as vitiligo,dermal melanocytosis, Franceschetti-Jadassohn Syndrome, etc. For suchdepigmentation applications, the formulation and dosing would be asdescribed above with respect to pigmentation applications.

Discovery of the pathway through which the present compounds act alsoleads to methods for screening compounds for melanogenic activity andpotency, or for their ability to reduce or suppress melanogenesis, basedon measurement of generation of nitric oxide (NO) or measurement ofnitric oxide synthesis (NOS) activity. Methods for measurement of NO orNOS include but are not limited to the following well known methods.Measurement of NO is usually based on the fact that NO rapidlydecomposes to nitrate and nitrite in aqueous solution. Nitrate reductaseis added to culture media or cell extracts to ensure complete conversionof nitrate to nitrite. Griess reagents (sulfanilamide andN-[1-naphthyl]-ethylenediamine) are then added to convert nitrite into adeep purple azo compound that absorbs maximally at 540 nm (Schmidt, etal., 1995, Biochemica 2:22). Reactions are typically carried out in a96-well format with absorbances read on a microtiter plate reader.Alternatively, following conversion of nitrate to nitrite as describedabove, DAN reagent (2,3-diaminonaphthalene) is added followed by NaOHwhich converts nitrite into the fluorescent compound1(H)-naphthotriazole. This is measured fluorimetrically with excitationat 365 nm and emission at 450 nm, typically in a 96-well format (Miles,et al., 1995, Methods 7:40). NOS activity is measured by adding[³H]-arginine to intact tissues or protein extracts, and measuringrelease of ³H resulting from the conversion of arginine to citrullineduring the enzymatic formation of NO by NOS (Baudouin and Tachon, 1996,J. Invest. Dermatol. 106:428-431). Alternatively, the production of cGMPor activity of PKG can be used as a screening tool. cGMP may be measuredby commercially available immunoassay (see Romero-Graillet, et al.,1996, J. Biol. Chem. 271:28052-28056). PKG may be measured by cyclic GMPdependent kination of a primary histone target (see Hidaka, et al.,Biochemistry 1984, 23, 5036-5041)

The use of and useful and novel features of the present methods andcompositions will be further understood in view of the followingnon-limiting examples.

EXAMPLE 1

The Cloudman S91 mouse melanoma cell line was obtained from AmericanType Culture Collection (ATCC). Cells were cultured in Dulbecco'sModified Eagles Medium (DMEM) containing 10% calf serum, 2 mML-glutamine, 10 U Penicillin/ml and 10 ug Streptomycin/ml according to apreviously published protocol (Eller, et al., Proc. Natl. Acad. Sci.93:1087-92. 1996). For testing propylene glycol and analogues forinduction of melanogenesis, S91 cells were plated at 10⁵ cells/35 mmdish in 10% calf serum. One day after plating, media was removed andreplaced with media containing 2% calf serum and test compounds (Eller,et al., 1996). Cells were cultured for 6 days at 37° C. in 5% CO₂ in ahumidified incubator. Following this treatment period, cells wereexamined microscopically and the portion of dedifferentiated anddifferentiated cells was estimated. Previous studies have shown thatdedifferentiated S91 cells have a rounded, spindly appearance whiledifferentiated S91 cells have a flattened, cuboidal, multipolar anddendritic appearance (Orlow, et al., Exp. Cell Res. 191:209-218, 1990).

Following this microscopic examination, cells were detached from dishesby trypsin. The time required for detachment by trypsin was recorded asan additional indicator of the phenotypic effects of test compounds. Foreach treatment, a subsample of cells was counted to determine theeffects of treatment compounds on cellular proliferation. The remainderof cells were used for determination of melanin content. Melanin wasextracted from cells by vortexing for 15 min in 1N NaOH. Standards wereprepared by dissolving melanin (Sigma) in 1 N NaOH (Eller, et al.,1996). Absorbance of standards and samples was measured at 475 nm.Melanin was expressed as pg melanin/cell.

Tables 1 and 2 below show the results obtained when testing formulationscontaining various concentrations of 1,2-propanediol as the activeingredient. In the control, no test compound was added to the medium.

TABLE 1 Concentration Cells (× 10⁶) ug Melanin pg Melanin/Cell Control0.48 2.52 5.3 1% (136 mM) 0.52 4.88 9.4 2% (272 mM) 0.50 6.24 12.5 3%(408 mM) 0.20 4.03 20.2 4% (544 mM) 0.10 4.01 40.1 5% (680 mM) 0.08 2.3128.9

TABLE 2 Morphology Rounded Flattened Trypsinization ConcentrationSpindly Cuboidal Detachment Time Control 100%  ≦3 min 1% (136 mM)  90% 10%  ≦6 min 2% (272 mM)  70%  30%  ≦9 min 3% (408 mM)  40%  60% ≦12 min4% (544 mM)  15%  85% ≦15 min 5% (680 mM) 100% ≦15 min

EXAMPLE 2

The same procedure as in Example 1 was followed, except that ethanol,and isomers of propanediol and butanediol were used as test compounds.The results are set forth in Tables 3 and 4. The data demonstrate thatseveral isomers of propanediol and butanediol induce melanogenesis anddifferentiation of S91 melanoma cells. Both 50 mM propanediol (PG) orbutanediol (BD) resulted in an approximate 1.5-fold increase ofmelanogenesis, while 150 mM resulted in about a 2-fold increasefollowing a single treatment. Whereas 1,2 propanediol (PG-1,2) and(S)-(+)-1,2-Propanediol (PG-S-1,2) resulted in no reduction of cellproliferation at the levels used in this experiment, 150 mM1,3-propanediol (PG-1,3), 2,3-butanediol (BD-2,3) or 1,3-butanediol(BD-1,3) resulted in a reduction of cell numbers by one-third. Inaddition, the butanediols appeared to result in greater differentiationof S91 cells than the propanediols, as evidenced by earlier and greatermorphological changes, and in the case of BD-2,3, a more adherentphenotype. Ethanol (EtOH) had no effect on cells at 340 mM but was toxicat 850 mM as indicated by low cell survival. Ethanol did not inducemelanogenesis at any concentration tested. Glycerol (G) had only aslight effect-on melanogenesis and differentiation at the concentrationstested in this experiment, indicating that triols may be less effectiveinducers of these phenotypes than diols.

TABLE 3 Cells (× 10⁶) ug Melanin pg Melanin/Cell Control 0.100 1.17 11.71.0% ETOH¹ 0.104 1.14 11.0 2.0% ETOH² 0.100 1.25 12.5 5.0% ETOH³ 0.0320.17 5.3  50 mM PG-1,2 0.084 1.31 15.6 150 mM PG-1,2 0.072 1.73 24.0  50mM PG-S-1,2 0.088 1.51 17.1 150 mM PG-S-1,2 0.080 2.04 25.5  50 mMPG-1,3 0.064 1.31 20.4 150 mM PG-1,3 0.044 1.04 23.6  50 mM G 0.092 1.0311.2 150 mM G 0.084 1.09 13.0  50 mM BD-2,3 0.072 1.12 15.6 150 mMBD-2,3 0.040 0.95 23.8  50 mM BD-1,3 0.064 0.99 15.5 150 mM BD-1,3 0.0480.87 18.1 ¹170 mM ²340 mM ³850 mM

TABLE 4 Morphology Rounded Flattened Trypsinization Spindly CuboidalDetachment Time Control 100% 3 min 1.0% ETOH 100% 3 min 2.0% ETOH 100% 3min 5.0% ETOH 100% 3 min  50 mM PG-1,2  75%  25% 3 min 150 mM PG-1,2 50%  50% 6 min  50 mM PG-S-1,2  75%  25% 3 min 150 mM PG-S-1,2  50% 50% 6 min  50 mM PG-1,3  75%  25% 3 min 150 mM PG-1,3  50%  50% 6 min 50 mM G 100% 3 min 150 mM G  75%  25% 3 min  50 mM BD-2,3  25%  75% 3min 150 mM BD-2,3 100% 9 min  50 mM BD-1,3  25%  75% 3 min 150 mM BD-1,3100% 6 min

Melanogenesis is the most characteristic feature of melanocytedifferentiation (J. Cell Sci. 107:1095-1103, 1994), and, is inverselycorrelated with rate of proliferation in melanoma cell lines (Neoplasia31:545-9, 1984; Biochem. Biophys. Res. Commun. 177:545-50, 1991; Exp.Dermatol. 4:192-198, 1995). As a general rule, increased proliferationcommensurate with dedifferentiation are hallmarks of rapid tumorprogression and a poor prognosis, while decreased proliferation anddifferentiation are indicative of more long-term survival (Introductionto the Cellular and Molecular Biology of Cancer, L. M. Franks and N.Teich, 1987, Oxford University Press). Thus, the ability of the presentcompounds to induce melanogenesis and slow cell growth is indicative oftheir ability to act as chemotherapeutic agents. Induction ofmelanogenesis combined with a reduced rate of cellular proliferation isindicative of induction of differentiation in S91 cells. In addition,the change of cellular morphology from a rounded, spindly appearance toa flattened, cuboidal appearance is further indication ofdifferentiation in S91 cells (Exp. Cell Res. 191:209-218, 1990). Thus,the compounds of the present invention are not only tanning agents, butalso chemotherapeutic agents capable of delaying tumor progression andincreasing long-term survival.

It should be noted that the effects of propylene glycol (Example 1) andrelated diols and triols (Examples 1 & 2) on S91 cells are identical tothose resulting from treatment of S91 cells with retinoids; that is,induction of melanogenesis, induction of differentiation, increasedadherence, and inhibition of proliferation (Laukharanta, et al., Arch.Dermatol. Res. 277:147-150, 1985). Given this similarity of biologicalresponses, it is believed that the agents described herein are effectivein treating those disorders presently treated with the retinoidsincluding a variety of forms such as psoriasis, acne and dermatoses.

EXAMPLE 3

The same procedures as in Examples 1 and 2 were followed to examine theeffect of additional compounds on melanogenesis in S91 cells. Theresults described in Table 5 show the concentration of a number ofcompounds required to induce 2-fold or greater melanization in S91cells. Many compounds are more potent than those described in Examples 1and 2. For example, 2,3-pyridinediol was potent at 100 uM;1,4-dioxane-2,3-diol and β-estradiol at 500 um;5-norbornene-2,2-dimethanol at 5 mM; 3,3-dimethyl-1,2-butanediol and1,2-cis-cyclopentanediol at 10 mM; and 2,3-dimethyl-2,3-butanediol at 25mM. All of the compounds listed in Table 5 except 1,4-dioxane-2,3-diol,induced transformation of S91 cells from a rounded bipolar morphology toa flattened cuboidal multipolar morphology concomitant with induction ofmelanogenesis; this indicates their potential usefulness aschemotherapeutic agents that act by inducing differentiation of tumorcells. All of the compounds listed in Table 5 except5-norbornene-2,2-dimethanol, β-estradiol, and 2,3-pyridinediol inducedincreased trypsinization time concomitant with induction ofmelanogenesis; alterations of adherence properties are related tochanges of metastatic potential of tumor cells.

TABLE 5 Concentration Required for ≧2-fold Compound Melanin Induction inS91 Cells 2,3-Pyridinediol 100 uM 1,4-Dioxane-2,3-Diol 500 uMβ-Estradiol 500 uM 5-Norbornene-2,2-Dimethanol  5 mM1,2-cis-Cyclopentanediol  10 mM 3,3-Dimethyl-1,2-Butanediol  10 mM2,3-Dimethyl-2,3-Butanediol  25 mM 1,2-trans-Cyclopentanediol  50 mM2-Methyl-1,3-Propanediol  50 mM 2,3-Butanediol 100 mM 1,2-Propanediol150 mM

Compounds in addition to those described in Examples 1 and 2, that didnot induce significant (≧2-fold increase) melanogenesis in S91 cellswhen tested over a range of concentrations up to a toxic dose included:1-propanol; 2-propanol; oleic acid; 2-phenyl-1,2-propanediol;1,3-cyclohexanediol; tartaric acid; ascorbic acid; Azone®,2-pyrrolidone; D-ribose; 2-deoxy-D-ribose; N-methyl-D-glucamine;hydroxymethyl uracil; and tetrabutylammonium chloride. Of thesecompounds, only 2-pyrrolidone resulted in profound morphologicaldifferentiation of S91 cells, indicating that it may augmentmelanogenesis and/or exert antitumorigenic activity in the absence ofmelanogenesis.

The PKC inhibitors H7 (1-[5-isoquinolinyl-sulfonyl]-2-methyl-piperazine)and D-sphingosine also induced melanogenesis in S91 cells. In addition,these PKC inhibitors enhanced melanogenesis induced by propylene glycolin S91 cells. These results indicate that propylene glycol does notinduce melanogenesis by induction of PKC, or require PKC for inductionof melanogenesis.

EXAMPLE 4

Normal human epidermal melanocytes (NHEMs) were examined for inductionof melanogenesis using cells and media from Clonetics Corporation (SanDiego, Calif.). Cells were cultured exactly as specified by thesupplier. Based on induction of a 1.5-fold increase of melanin in NHEMs,the most potent compound examined was 2,3-pyridine-diol at 200 uM,followed by 5-norbornene-2,2-dimethanol at ≦5 mM,3,3-dimethyl-1,2-butanediol at 12.5 mM, and 2,3-dimethyl-2,3-butanedioland 1,2-cis-cyclopentanediol at 50 mM (Table 6). D-Ribose was inactivein NHEMs when tested over a range of concentrations up to a toxic dose.These results show that compounds of the present invention that exhibitactivity in S91 cells, also exhibit activity in normal humanmelanocytes.

TABLE 6 Concentration Required for ≧1.5-fold Compound Melanin Inductionin NHEMs 2,3-Pyridinediol  200 uM 5-Norbornene-2,2-Dimethanol   5 mM3,3-Dimethyl-1,2-Butanediol 12.5 mM 1,2-cis-Cyclopentanediol   50 mM2,3-Dimethyl-2,3-Butanediol   50 mM 1,2-Propanediol  150 mM

EXAMPLE 5

Compounds were tested for melanogenic activity in vivo by application toAmerican short-haired guinea pigs. Treatment sites were created byremoval of fur using Nair® brand depilatory. Compounds were applied in25 μl volumes twice per day for 5 days to each treatment spot asindicated in Table 7. In the Table, the numbers presented are therelative melanogenesis rating (mean±SE), and are arranged according tothe relative location on the animal, with the head being to the left andthe tail being to the right. Propylene glycol (PG=13.6M), 2,3-butanediol(2,3-BD=10.95M), and 1,2-cis-cyclopentanediol (1,2-cs-CPD=10.7M) wereapplied as full strength solutions. 3,3-dimethyl-1,2-butanediol(3,3-M-1,2-BD) was applied as a 4M solution dissolved in ethanol. Twoweeks following cessation of treatments, the degree of pigmentation wassubjectively rated according to the following scale:

0 no change 0.5 slight darkening, not easily discernible 1 slightdarkening, easily discernible 2 moderate, even darkening 3 substantial,even darkening 4 profound, even darkening

The results presented below showed that there was a progressivediminution of response to tanning agents from head to tails of animals.The magnitude of this diminished response was 3- to 4-fold. Thus,comparisons between treatment compounds were done relative to similarlocations on the body of guinea pigs. Propylene glycol resulted insignificant melanogenesis relative to depilitory treated controlslocated at the same relative body position. 2-methyl-1,3-propyleneglycol and 2,3-butanediol were only slightly better melanogenic agentsthan propylene glycol. However, 3,3-dimethyl-1,2-butanediol and1,2-cis-cyclo-pentanediol resulted in 4.5-fold and 5.5-fold greatermelanogenesis than PG applied at similar body locations.

TABLE 7 Treatment Head<--------------------------------------------------------> Tail a b c dPG, 5 Days (n = 6): 1.04 ± 0.21 0.83 ± 0.17 0.25 ± 0.09¹ 0.33 ± 0.16¹ 5days (n = 3): 2-M-PG 2,3-BD 2-M-PG 2,3-BD 1.25 ± 0.52 1.33 ± 0.17² 0.58± 0.08² 0.25 ± 0.14 5 Days (n = 3): Nair PG 3,3-M-1,2-BD 1,2-cs-CPD 0²0.50 ± 0.25 1.16 ± 0.66² 1.83 ± 0.33² ¹P < 0.05 relative to PG-treatedsite located nearest head in first row ²P < 0.05 relative to PG-treatedsite in first row that is located at same position relative to head andtail

In order to minimize the effects of dimunition of response from head totails of animals, all future experiments were done using only treatmentspots located towards the tails of animals (c and d in Table 7). Deemedas additionally beneficial, in this area of the animal differences ofresponsiveness to strong and weak inducers of pigmentation, as deducedfrom cell culture, were greatest. Comparison of the pigmentation ratingsof these treatment spots showed the following descending order ofinduction: 8.7M 1,2-cis-cyclopentanediol (1,2-cs-CPD)>4M3,3-di-methyl-1,2-butanediol (3,3-M-1,2-BD)>a mixture of 8.5M1,2-propylene glycol (1,2-PG)/1M 5-norbornene-2,2-dimethanol(5-NBene-2,2-DM)/2% 2-pyrrolidone (2-P; a penetration enhancer)>1M5-NBene-2,2-DM/2% 2P,>11.3M 2-methyl-1,3-propylene glycol (2-M-1,2-PG)(Table 8; FIG. 1A: untreated; 1B: 10.6M 1,2-PG/2% 2-P; 1C: 8.7M1,2-cs-CPD; 1D: 1M 5NBene-2,2-DM/8.5M 1,2-PG/2% 2-P). In this region ofthe animals, responses to 13.61M 1,2-PG; 10.6M 1,2-PG/2% 2P, and 11M2,3-dimethyl-2,3-butanediol were not significantly different fromcontrol (Nair or 2% 2P treated) spots. Pigmentation ratings werecorrected for background (control treatment spots), normalized to 1M toaccount for the different amounts of each agent applied, and thennormalized to results for 1,2-PG (Table 8). This comparison showed thatthe descending order of induction was5-NBene-2,2-DM>1,2-cs-CPD>2-M-1,3-PG, and, that using 1,2-PG as carrierfor 5-NBene-2,2-DM (FIG. 1D) increased responsiveness to this compound.It is anticipated that further improvements in formulation willadditionally improve responsiveness to 5-NBene-2,2-DM and othercompounds in this invention. Biopsies results (FIG. 1) showed thatinduction of melanogenesis was marked by deposition of melanin inkeratinocytes, in some cases with formation of “supranuclear caps”(arrows, FIGS. 1C & 1D) indicative of induction of true naturalUV-protective melanogenesis (Gates, R. R., and A. A. Zimmerman, 1953 J.Invest. Dermatol. 21:339-348), and a complete absence of inflammation,fibrosis or any other form of tissue damage.

TABLE 8 Pigmentation Background Normalized Normalized Treatment RatingCorrected to 1M to 1,2-PG No Penetration Enhancer Nair 0.08 ± 0.05 0 (n= 6) 13.61M 0.29 ± 0.09 0.21 ± 0.03 0.015 ± 0.002 1.0 ± 0.1 1,2-PG  (n =12) 11.0M 0.25 ± 0.14 0.17 ± 0.09 0.015 ± 0.008 1.0 ± 0.6 2,3-M-2,3-BD(n = 3) 11.3M  0.58 ± 0.08* 0.50 ± 0.07 0.044 ± 0.006 2.9 ± 0.42-M-1,3-PG (n = 3) 8.7M  1.89 ± 0.27* 1.75 ± 0.25 0.202 ± 0.029 13.5 ±1.9  1,2-cs-CPD (n = 9) 4.0M  1.17 ± 0.44* 1.09 ± 0.41 0.272 ± 0.10218.1 ± 6.8  3,3-M-1,2-BD (n = 3) Penetration Enhancer 2% 2-Pyrrolidone2P 0.17 ± 0.08 0 (n = 6) 10.6M 0.33 ± 0.05 0.16 ± 0.02 0.015 ± 0.002 1.0 ± 0.15 1,2-PG/2P (n = 6) 1.0M  0.66 ± 0.05* 0.49 ± 0.04 0.490 ±0.037 32.7 ± 2.5  5-NBene-2,2-DM/2P (n = 6) 8.5M  1.00 ± 0.13* 0.83 ±0.11  0.670 ± 0.087¹ 44.7 ± 5.8  1,2-PG/2P/1.0M 5-NBene-2,2-DM (n = 6)*P < 0.05; Students T-test ¹Further background corrected forpigmentation induced by 1,2-PG/2P (0.16)

EXAMPLE 6

Compounds were examined for their ability to induce tyrosinase activityin S91 mouse melanoma cells. Tyrosinase is the rate limiting enzyme inthe melanogenic pathway. Its measurement provides a highly specific andsensitive indication of degree of induction of melanogenesis by testcompounds. All cell culture conditions and treatments were as describedabove in Examples 1-3. Following treatments, cells were trypsinized,counted by Coulter, pelleted by centrifugation at 1000×g, and analyzedfor tyrosinase activity using modifications of previously describedprocedures (Pomerantz, S. H., 1966, J. Biol. Chem. 241:161-168; Jara, etal., 1988, Pigment Cell Res. 1: 332-339.). Briefly, cell pellets weresolubilized by sonicating for 5 seconds in 600 ul 50 mM phosphate bufferpH 6.8 containing 0.5% Triton-X100, followed by vortexing, incubation onice for 30 min, and then revortexing. From this, 200 ul aliquots werecombined with 200 ul of reaction mixture containing either 75 uMtyrosine, 75 uM L-Dopa, and 2 uCi L-[3,5- ³H]Tyrosine in 50 mM NaPO₄ pH6.8 (L-Dopa+), or, 75 uM tyrosine, and 2 uCi L-[3,5- ³H]Tyrosine in 50mM NaPO₄ pH 6.8 (L-Dopa−) and incubated 1 hr at 37° C. Reactions werestopped by addition of 400 ul 10% activated charcoal in 0.1N HCl andincubation on ice for 15 min. This mixture was centrifuged at 17,300×gfor 5 min, and 400 ul supernatant was then filtered through a 0.22 uM GVDurapore centifugal filter unit (Millipore) by centrifuging at 17,300×gfor 5 min. Filtrate was added to 4 ml Fisher Plus scintillation fluidand counted on a Hewlett Packard scintillation counter. Tyrosinaseactivity was calculated as dpm/hr/ug protein and dpm/hr/10³ cells. Eachsample was analyzed with and without L-Dopa, a necessary cofactor fortyrosinase (Pomerantz, S. H., 1966, J. Biol. Chem. 241:161-168; McLane,et al., 1987, Biochem. Biophys. Res. Commun. 145:719-725). All reportedtyrosinase values are exclusive of counts that occurred in buffer blanksand L-dopa negative aliquots. Protein was determined on aliquots of celllysate, extracellular particulate lysate or media by the BradfordCoomassie Blue method (Bradford, 1967, Anal. Biochem. 72:248-254) usingBio-Rad Protein Assay Kit I.

Results (Table 9; mean±SE) show that 3,3-dimethyl-1,2-butanediol(3,3-M-1,2-BD) and 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM) resultin the greatest induction of tyrosinase on both a cellular and proteinbasis. Although 100 uM 2,3-pyridinediol (2,3-Pyd) induced 2-foldincreases of melanin (Example 3, Table 5), even 500 uM 2,3-Pyd inducedonly low levels of tyrosinase relative to that induced by 5 mM5-NBene-2,2-DM or 3,3-M-1,2-BD, and, higher levels of 2,3-Pyd weretoxic. 5-NBene-2,2-DM and 3,3-M-1,2-BD are nontoxic at concentrationsthat induce much higher levels of tyrosinase, and thus are preferredagents for induction of melanogenesis in this embodiment. Since5-NBene-2,2-DM induces nearly equivalent levels of tyrosinase at 5-foldlower concentrations than 3,3-M-1,2-BD, it is particularly preferred.IBMX (3-isobutyl-1-methylxanthine) is well known to those in the art aspotent inducer of melanogenesis and tyrosinase, and is provided as apositive control.

TABLE 9 dpm/hr dpm/hr ug Sample #/Treatment 10³ Cells Protein Control (n= 4)  40 ± 6  184 ± 27 300 mM PG-1,2 (n = 4)  292 ± 104 1003 ± 370  25mM 3,3-M-1,2-BD (n = 2) 1211 ± 38 1746 ± 220  50 mM 1,2-cs-CPD (n = 2) 276 ± 16  925 ± 53  5 mM 5-NBene-2,2-DM (n = 4)  707 ± 54 1643 ± 105 0.5 mM 2,3-Pyd (n = 2)  142 ± 8  160 ± 19  0.1 mM IBMX (n = 2)  765 ±53 2161 ± 41

Structure activity studies with 5-NBene-2,2-DM and related compoundsindicate that norbornane-2,2-dimethanol (NBane-2,2-DM) has equivalentpotency for induction of tyrosinase in S91 cells (FIG. 2). Thus,NBane-2,2-DM is equivalently preferred with 5-NBene-2,2-DM. Lesserinduction of tyrosinase in S91 cells was induced in descending order by2-Norbornanemethanol (2-NBaneM), 2,3-cis/exo-Norbornanediol(2,3-c/e-NBaneD), α-Norborneol (α-NBane-ol), and Norbornane (NBane).Since even NBane results in 2-fold induction of tyrosinase relative tountreated or ethanol (ETOH) treated control S91 cells, it is included asa component of this invention. In addition, since NBane inducesmelanogenesis, it is contemplated that all compounds containing NBane asa component of their structure may induce melanogenesis. In addition,compounds containing Norbornene (NBene) or any other unsaturatedcompound derived form norbornane are expected to induce melanogenesis.Thus, any saturated or unsaturated compound derived from or related tonorbornane is included as a component of this invention, including butnot limited to compounds derived from bornane, pinane, camphene andcamphor.

Neither the highly specific protein kinase A (PKA) inhibitor H-89(N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfinamide.2HCl;Chijiwa, et al., 1990, J. Biol. Chem. 265:5267-5272), nor the highlyspecific protein kinase C (PKC) inhibitor GF109203X(Bisindolylmaleimide; Toullec, et al., 1991, J. Biol. Chem.266:15771-15781) inhibited induction of tyrosinase by 5-NBene-2,2-DM(Table 10). Thus, similar to results described for 1,2-propanediol inExample 3, 5-NBene-2,2-DM and related compounds are unlikely to act viaactivation of PKC pathways, which have been described as important forinduction of melanogenesis by diacylgerols (Allan, et al., 1995, J.Invest. Dermatol. 105:687-692; Gilchrest, et al., 1996, Photochem.Photobiol. 63:1-10). Nor are 5-NBene-2,2-DM or related compounds likelyto act via activation of PKA pathways, described as important forinduction of melanogenesis by IBMX (Fuller, et al., 1993, Ann. NY Acad.Sci. 690:302-319; Fuller, et al., 1996, Pigment Cell Res. S5:65).Furthermore, addition of catalase to the cell culture media did notinhibit the action of 5-NBene-2,2-DM, indicating that unlike L-Dopa andDopac, this and related compounds are unlikely to induce melanogenesisvia generation of hydrogen peroxide or other reactive oxygen species(Karg, et al., 1989, Acta Derm. Venereol. 69:521-524; Karg, et al.,1991. J. Invest. Dermatol. 96:224-227; Karg, et al., 1993, J. Invest.Dermatol. 100:209S-213S).

TABLE 10 Tyrosinase dpm/hr/ug Relative to Protein Control Control 398 15 mM 5-NBene-2,2-DM 3273 8.2X 1 uM H-89 507 1.3X 10 uM H-89 1236 3.1X 1uM H-89/ 4624 11.6X 5 mM 5-NBene-2,2-DM 10 uM H-89/ 3093 7.8X 5 mM5-NBene-2,2-DM 0.1 uM GF109203X 1025 2.6 1 uM GF109203X 2407 6.1X 0.1 uMGF109203X/ 4679 11.8X 5 mM 5-NBene-2,2-DM 1 uM GF109203X/ 6531 16.4X 5mM 5-NBene-2,2-DM 500 Units Catalase/ml 745 1.9X 1000 Units Catalase/ml691 1.7X 500 Units Catalase/ml/ 2796 7.0X 5 mM 5-NBene-2,2-DM 1000 UnitsCatalase/ml/ 4778 12.0X 5 mM/5-NBene-2,2-DM

EXAMPLE 7

Tyrosinase was measured in normal human epidermal melanocytes (NHEM)using procedures identical to those described for S91 cells (Example 6),except that media from day treatment periods was retained andcentrifuged at 200×g, 1600×g, or 17,300×g for analysis of tyrosinaseactivity in the extracellular exported melanosomal particulate fraction,and in the resultant supernatant media fraction. In some cases (Table11), tyrosinase was also measured by an in situ assay whereinradiolabelled tyrosine was added directly to freshly replaced media ofNHEM for a period of 24 hrs following a 5 day treatment period(Abdel-Malek, et al., 1992, J. Cell. Physiol. 150:416-425). Resultsshowed that 5 mM 5-NBene-2,2-DM induced tyrosinase to a greater extentin the in situ assay, in cells, in extracellular particulate melanosomalfractions, and in the media of NHEM than did 25 mM 3,3-M-1,2-BD (Table11). Both 5 mM 5-NBene-2,2-DM and 25 mM 3,3-M-1,2-BD induced moretyrosinase in each of these assays and fractions than did 1,2-PG. IBMX(3-isobutyl-1-methyl-xanthine) provided as a positive control, inducedas much tyrosinase as 5 mM 5-NBene-2,2-DM in the in situ assay, but lessin cellular, extracellular particulate and media fractions (Table 11).

TABLE 11 Tyrosinase dpm/hr/10³ Cells In 200 g 17300 g* Situ CellularPartic Partic Media** Control 16.8 10259  244  97 1457 85 mM ETOH 15.010201  442 132 1654 (1.00X) (1.00X) (1.00X) (1.00X) (1.00X) 300 mM1,2-PG 16.8 10247  433 102 1864 300 mM 1,2-PG 17.2 10875  923 241 2123(1.07X) (1.03) (1.98X) (1.50X) (1.28X) 25 mM 20.5 11728 1646 536 54953,3-M-1,2-BD 25 mM 21.0 11730 2226 425 3056 3,3-M-1,2-BD (1.31X) (1.15X)(5.64X) (4.20X) (2.75X) 5 mM 24.5 13838 6447 493 4164 5-NBene-2,2-DM 5mM 25.4 14716 6291 473 4639 5-NBene-2,2-DM (1.57X) (1.40X) (18.6X)(4.22X) (2.83X) 0.1 mM IBMX 25.3 10910 2189 220 2698 0.1 mM IBMX 26.111737 1834 260 2935 (1.62X) (1.11X) (5.86X) (2.10X) (1.81X) *Post 200 Xg **post 17300 X g

Further studies using NHEM demonstrated that, similar to results for S91cells (FIG. 2), compounds related to 5-NBene-2,2-DM may be inducers oftyrosinase (Table 12). For example, 2-norbornanemethanol (2-NBaneM)resulted in induction of tyrosinase at levels equivalent to5-NBene-2,2-DM in NHEM both from a white adult donor and a blackneonatal donor (Table 12). Thus, similar to S91 cells (Example 6), allnorbornane-related compounds are contemplated to induce tyrosinase inNHEM and are thereby embodied in this invention.

TABLE 12 Tyrosinase dpm/hr/10³ cells White-Adult-NHEM In Situ CellularMedia¹ Control 5.56 (1.00X) 13992 (1.00X) 36.3 (1.00X) 1 mM5-NBene-2,2-DM 6.27 (1.13X) 12740 (0.91X) 29.9 (0.82X) 5 mM5-NBene-2,2-DM 5.81 (1.04X) 18467 (1.32X) 53.1 (1.46X) 1 mM 2-NBaneM7.05 (1.27X) 15257 (1.09X) 29.2 (1.11X) 5 mM 2-NBaneM 6.18 (1.11X) 16077(1.15X) 48.1 (1.33X) dpm/hr/10³ cells Black-Neonatal-NHEM In SituCellular Media Control 12.5 (1.00X)  9856 (1.00X) 11.1 (1.00X) 1 mM5-NBene-2,2-DM 13.9 (1.11X) 10679 (1.08X) 26.8 (2.41X) 5 mM5-NBene-2,2-DM 14.1 (1.13X) 15398 (1.56X) 33.2 (2.99X) 1 mM 2-NBaneM12.1 (0.97X) 10863 (1.10X) 18.7 (1.68X) 5 mM 2-NBaneM 12.8 (1.02X) 17397(1.77X) 37.3 (3.36X) ¹Unlike Table 11 where Media was from a 5 daytreatment period, Media in Table 12 was from a 1 day treatment period.

EXAMPLE 8

Similar to results for S91 cells treated with diols (Examples 1 and 2),treatment of normal human epidermal melanocytes (NHEM) with 5 mM5-NBene-2,2-DM resulted in morphological changes indicative ofdifferentiation. In the case of NHEM, induction of differentiation wasmarked by conversion of cells from a bipolar phenotype to amultidendritic phenotype (compare untreated NHEM in FIG. 3A with 5 mM5-NBene-2,2-DM treated NHEM in FIG. 3B). Additionally, the length ofdendrites was increased approximately 2-3-fold following treatment with5 mM 5-NBene-2,2-DM, and there was an increase in the number ofsecretatory vesicles at the termini of dendrites (arrows in FIGS. 3A and3B). Electron microscopic analysis indicated that the extracellularparticulate fraction secreted into the media from NHEM was comprisedalmost exclusively of stage III and IV melanosomes (arrows showlongitudinal view and arrowheads show cross-sectional view in FIGS. 3Cand 3D). Increased secretion of melanosomes resulting from treatmentwith 5 mM 5-NBene-2,2-DM was reflected in increased extracellularparticulate tyrosinase activity (Example 7, Table 11).

It is well known that ultraviolet irradiation of skin results inincreased dendricity of melanocytes and increased transport ofmelanosomes from the ends of dendritic processes to neighboringkeratinocytes (Jimbow, et al., Biology of Melanocytes, pp. 261-289, In:Dermatology in General Medicine, eds: Fitzpatrick, et al., McGraw-Hill,1994). Thus, secretion of melanosomes from melanocytes treated with5-NBene-2,2-DM appears to parallel the physiological processes inducedby sunlight in skin.

EXAMPLE 9

Highly specific inhibitors of the cAMP/PKA (protein kinase A) or PKC(protein kinase C) pathways do not inhibit induction of melanogenesis by5-NBene-2,2-DM in S91 cells (Example 6, Table 10). However, each of thenitric oxide (NO) scavenger PTIO(2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide), the cyclicguanosine monophosphate (cGMP) inhibitor LY83583(6-anilino-5,8-quinolinequinone), and the PKG (protein kinase G)inhibitor KT58223 reduce induction of melanogenesis by 5-NBene-2,2-DM inS91 cells (Table 13). These results demonstrate that induction ofmelanogenesis by 5-NBene-2,2-DM occurs by the NO/cGMP/PKG pathway.Furthermore, results are similar to those obtained for ultravioletradiation wherein induction of melanogenesis did not occur via eitherthe cAMP/PKA or PKC pathways (Friedmann and Gilchrest, 1987, J. Cell.Physiol 133:88-94; Carsberg, et al., J. Cell. Sci. 107:2591-2597), butrather occurred via the NO/cGMP/PKG pathway (Romero-Graillet, et al.,1996, J. Biol. Chem. 271:28052-28056; Romero-Graillet, et al., 1997, J.Clin. Invest. 99:635-642). Moreover, unlike IBMX(3-isobutyl-1-methylxanthine) and MSH (melanocyte stimulating hormone)which induce melanogenesis by the cAMP/PKA pathway (Wintzen andGilchrest, 1996, J. Invest. Dermatol. 106:3-10; Fuller, et al., 1993,Ann. NY Acad. Sci. 690:302-319), and DAG (diacylglycerol) which inducesmelanogenesis by the PKC pathway (Allan, et al., 1995, J. Invest.Dermatol. 105:687-692), 5-NBene-2,2-DM induces melanogenesis by theNO/cGMP/PKG pathway similar to ultraviolet radiation.

It has been previously demonstrated that a variety of aliphatic andalicyclic diols including 5-norbornene-2,2-dimethanol (5-NBene-2,2-DM)induce melanogenesis in S91 cells (Examples 1-3). The results presentedin Table 15 show that induction of tyrosinase (the rate-limiting enzymein melanogenesis) by 5-NBene-2,2-DM is not blocked by highly specificinhibitors of the PKC and PKA pathways. In fact, treatment of S91 cellswith either the highly specific PKA inhibitor H-89 (Chijiwa, et al.,1990, J. Biol. Chem. 265:5267-5272), or the highly specific PKCinhibitor GF109203X (Toullec, et al., 1991, J. Biol. Chem.266:15771-15781) resulted in augmentation of basal and5-NBene-2,2-DM-induced tyrosinase levels (Table 15). Thus,5-NBene-2,2-DM does not appear to act via either the PKC or PKApathways.

In contrast, both the nitric oxide (NO) scavenger PTIO(2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), the cyclicguanosine monophosphate (cGMP) inhibitor LY83583(6-anilino-5,8-quinolinequinone), and the PKG (cGMP-activated proteinkinase) inhibitor KT5823 reduced induction of melanogenesis by5-NBene-2,2-DM in S91 cells (Table 16). These results demonstrate thatinduction of melanogenesis by 5-NBene-2,2-DM occurs by the NO/cGMP/PKGpathway.

Previously, it has been demonstrated that NO donors can stimulatemelanogenesis in normal human melanocytes (Romero-Graillet, et al.,1996, J. Biol. Chem. 271). Results presented here demonstrate that5-NBene-2,2-DM can stimulate melanogenesis with an efficacy equivalentor greater than that of NO donors, even though 5-NBene-2,2-DM has noability to donate NO. Since induction of melanogenesis by 5-NBene-2,2-DMoccurs by the NO/cGMP/PKG pathway, 5-NBene-2,2-DM must directlystimulate NO synthesis within cells.

These results demonstrate that stimulation of NO synthesis and thecGMP/PKG pathway by 5-NBene-2,2-DM provides an efficient alternative tostimulation of this pathway by NO donors. Thus, 5-NBene-2,2-DM andrelated compounds described in this invention will serve as alternativetherapeutics for treatment of a variety of diseases mediated byperturbations of the NO/cGMP/PKG pathway.

TABLE 13 % of dpm/hr/ 5-NBene- Induction 10³ cells 2,2-DM NO/PKGInhibitors - Experiment 1 5 mM 5-NBene-2,2-DM (n = 4) 5018 ± 415¹ 100% 5mM 5-NBene-2,2-DM/ 3703 ± 262  74% 20 uM PTIO² (n = 2) 5 mM5-NBene-2,2-DM/ 1528 ± 190  31% 0.5 uM KT5823³ (n = 2) NO/PKGInhibitors - Experiment 2 5 mM 5-NBene-2,2-DM (n = 4) 5640 ± 323 100% 5mM 5-NBene-2,2-DM/ 4078 ± 429  72% 20 uM PTIO² (n = 2) 5 mM5-NBene-2,2-DM/ 3351 ± 994  59% 40 uM PTIO (n = 2) 5 mM 5-NBene-2,2-DM/2940 ± 261  52% 0.5 uM KT5823³ (n = 2) 5 mM 5-NBene-2,2-DM/ 1688 ± 324 30% 1.0 uM KT5823 (n = 2) cGMP Inhibitor - Experiment 3 5 mM5-NBene-2,2-DM (n = 4) 6388 ± 460¹ 100% 5 mM 5-NBene-2,2-DM/ 1389 ± 64 22% 0.1 uM LY83583⁴ (n = 2) 5 mM 5-NBene-2,2-DM/  300 ± 84  5% 0.2 uMLY83583 (n = 2) ¹X ± SE ²PTIO: Nitric oxide scavenger ³KT5823: PKGinhibitor ⁴LY83583: inhibitor of cGMP formation.

EXAMPLE 10

Studies with the L-arginine analog S-ethylisothiourea (Garvey, et al.,1994, J. Biol. Chem. 269:26669-26676; Southern, et al., 1995, Br. J.Pharmacol. 114:510-516), a competitive inhibitor of nitric oxidesynthase at the L-arginine binding site, also the contention that5-norbornene-2,2-dimethanol acts via the nitric oxide pathway. Treatmentwith S-ethylisothiourea (S-EITU) resulted in a dose-response diminutionof tyrosinase activity in S91 cells, with complete ablation oftyrosinase activity at 1000 nM S-EITU (Table 14).

TABLE 14 Tyrosinase dpm/hr/10³ Cells Control  47 86 mM ETOH  46 5 mM5-NBene-2,2-DM 4515 5 mM 5-NBene-2,2-DM 4247   Avg. 4381 5 mM5-NBene-2,2-DM/50 nM S-EITU 5186 5 mM 5-NBene-2,2-DM/100 nM S-EITU 46465 mM 5-NBene-2,2-DM/250 nM S-EITU 3758 5 mM 5-NBene-2,2-DM/500 nM S-EITU1055 5 mM 5-NBene-2,2-DM/750 nM S-EITU  357 5 mM 5-NBene-2,2-DM/1000 nMS-EITU Not Done

EXAMPLE 11

As a continuance of the structure activity studies described in Example6 (FIG. 2), a variety of norbornane derivatives and related monocyclicor aliphatic derivatives were examined for melanogenic activity (Tables15 and 16). Although many of these agents possessed significantmelanogenic activity, only 2-hydroxy-2-norbornanemethanol and 1-(exo &endo-2-norbornyl-)-propan-1,2-diol induced levels of tyrosinase thatapproached the maximal levels induced by 5-norbornene-2,2-dimethanol(Table 16).

The results presented in Table 16 demonstrate that several differenttypes of norbornane derivatives including triols, acetates, acetateesters, carboxylic acids, and formates possess melanogenic activity. Asshown previously in Example 6, some of this activity is embodied withinthe norbornane structure itself, since 1 or 5 mM norbornane resulted in2-fold induction of tyrosinase (FIG. 2). These results furthersubstantiate the claims herein, that any compound derived fromnorbornane is expected to be a melanogenic agent, and is thereforeincluded in this invention.

Low levels of melanogenic activity were also exhibited by monocyclicdimethanol compounds and a noncyclic dimethanol-containing compound(Table 16). These results demonstrate that dimethanol groups embody lowlevels of melanogenic activity, even in the absence of the bicyclic ringstructure of norbornane. These results, combined with the finding that2-norbornanemethanol exhibits significant melanogenic activity (FIG. 2;Table 12), demonstrate that any compound containing one or more methanolgroups has the potential to be a melanogenic agent, and these aretherefore also included in this invention.

TABLE 15 Fold Induction of Tyrosinase Relative to Controls 1 mM 2 mM 5mM 5-norbornene-2,2-dimethanol 4.0X 12.9X 41.9X 2,5 &6,7-norbornanetriol¹ 5.1X  5.1X  ND² mono- & di-acetate 1.5X  3.0X ND2,5 & 6,7-norbornanetriol 2-norbornaneacetic acid 4.6X ND 12.9X5-norbornene-2,3-cis/endodicarboxylic 2.2X ND  1.0X acid ±exo-2-norbornyl formate 2.7X ND  2.3X ¹5 & 6 refers to a mixture ofmolecular entities wherein hydroxyl substituents may be in either the 5or 6 position ²ND: not done

TABLE 16 Fold Induction of Tyrosinase Relative to Controls 0.5 mM 1 mM2.5 mM 5 mM 5-norbornene-2,2-dimethanol 2.8X 3.6X 14.5X 61.6X2,7-norbornanediol ND¹ 0.8X 1.6X  3.5X 2-hydroxy-2-norbornanemethanol ND9.2X 15.5X 45.0X 1-(exo & endo-2-norbornyl-)- 2.2X 4.2X 48.5X  2.0Xpropan-1,2-diol methyl-5-norbornene-2,3- 6.0X 8.1X 0.8X  NA² dimethanol1,2-cis-cyclohexanedimethanol ND 0.9X 6.1X NA3-cyclohexane-1,1-dimethanol ND 1.4X 2.3X  7.0X1,4-cyclohexanedimethanol ND 1.2X 1.7X  3.2X pentaerylthritol ND 1.2X1.9X  1.5X ¹ND: not done ²NA: not analyzed because cells had detachedfrom culture dishes

EXAMPLE 12

Further studies using S91 cells and the methods described in Example 6showed that 2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol) hadgreater melanogenlc activity than 5-norbornene-2,2-dimethanol whentested over a range of concentrations (FIG. 5). 2,3-cis/exo-pinanediolinduced 2.6-fold more tyrosinase activity than5-norbornene-2,2-dimethanol when tested at 500 uM, 5.2-fold more at 1mM, and 7.3-fold more at 2.5 mM (calculated from data in FIG. 5).

In a related experiment, nitric oxide was measured in cell-free mediafrom S91 cells following treatment with a range of concentrations of2,3-cis/exo-pinanediol or 5-norbornene-2,2-dimethanol for 4 days. Inbiological fluids, nitric oxide is converted into nitrite and nitratewith seconds of production. Therefore, nitric oxide is measured by firstconverting nitrate to nitrite using nitrate reductase, followed byaddition of Greiss reagent to detect nitrite as optical density at 550nm (Moshage, et al., 1995, Clin. Chem. 41:892-896; Schmidt, et al.,1995, Biochemica 2:22)). Results of this experiment showed that2,3-cis/exo-pinanediol is a more potent inducer of nitric oxidesynthesis than 5-norbornene-2,2-dimethanol (Table 17). Moreover, therelative melanogenic potency of 2,3-cis/exo-pinanediol and5-norbornene-2,2-dimethanol shown in FIG. 5 paralleled the relativepotency of these compounds with regards to induction of nitric oxide(Table 17). These results in combination with those given in Example 9indicate that similar to induction of melanogenesis by ultravioletirradiation (Romero-Graillet, et al., 1996, J. Biol. Chem.271:28052-28056; Romero-Graillet, et al., 1997, J. Clin. Invest.99:635-642), induction of melanogenesis by diols occurs via the nitricoxide pathway. It follows that measurement of induction of nitric oxide,cGMP or PKG may provide biochemically relevant screening assays forcompounds that may be melanogenic. Thus, the utilization of these assaysto screen compounds for melanogenic activity is claimed in the presentinvention.

In addition to being a more potent inducer of melanogenesis and nitricoxide (FIG. 4 and Table 17), 2,3-cis/exo-pinanediol was also a morepotent inducer of cell cycle arrest than 5-norbornene-2,2-dimethanol(Table 17). As discussed in Example 2, induction of melanogenesis inassociation with cell cycle arrest is indicative of inductiondifferentiation of melanoma cells. This indicates that2,3-cis/exo-pinanediol may have even greater utility than5-norbornene-2,2-dimethanol for use as a chemotherapeuticdifferentiation agent for treatment of melanoma and other types ofcancers.

TABLE 17 nmoles NO/ Cells (× 10⁶) uM NO 10⁶ Cells Untreated 0.409 ±0.037 1.74 ± 0.39 4.36 ± 1.16   1 mM 5-NBene-2,2-DM¹ 0.423 ± 0.052 4.80± 0.32 11.6 ± 1.1* 2.5 mM 5-NBene-2,2-DM 0.269 ± 0.040* 5.46 ± 0.32 21.4± 3.7*   5 mM 5-NBene-2,2-DM 0.090 ± 0.011* 6.36 ± 0.12 72.9 ± 10.5* 0.5mM 2,3-cs/ex-PD² 0.325 ± 0.002 3.54 ± 0.06 10.9 ± 0.2*   1 mM2,3-cs/ex-PD 0.258 ± 0.010* 6.36 ± 1.56 24.5 ± 5.4* 2.5 mM 2,3-cs/ex-PD0.099 ± 0.014* 12.6 ± 0.5  131 ± 15*   5 mM 2,3-cs/ex-PD 0.064 ± 0.006*11.0 ± 1.1  174 ± 15*  85 mM ETOH³ 0.454 ± 0.036 3.18 ± 0.49 7.04 ± 1.00¹5-NBene-2,2-DM: 5-norbornene-2,2-dimethanol ²2,3-cs/ex-PD:2,3-cis/exo-pinanediol ([1R,2R,3S,5R]-[−]-pinanediol) ³ETOH: ethanolsolvent control for 5 mM 5-NBene-2,2-DM and 5 mM 2,3-cs/ex-PD (lowertreatment concentrations received proportionally less ETOH) *p < 0.05;T-test (X ± SE; n = 3)

EXAMPLE 13

In studies using the guinea pig model identical to that described inExample 5, 2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol)exhibited 2- to 4-fold more melanogenic activity than equivalentconcentrations of 5-norbornene-2,2-dimethanol when compared usingtreatment spots in the posterior half of animals (c and d in Table 18and FIG. 5). In FIG. 5, a, b, c and d indicate treatment spots thattransverse the anterior-posterior axis along the backs of guinea pigs.FIG. 5A, top row, shows spots a-d treated with 50% ETOH; FIG. 5A, bottomrow, shows spots a-d treated with 8.7M 1,2-cis-cyclopentanediol in 20%ETOH; FIG. 5B, top row, shows spots a-d treated with 1M5-norbornene-2,2-dimethanol in 8.5M propylene glycol, 20% ETOH, and 2%2-pyrrolidone; and FIG. 5B, bottom row, shows spots a-d treated with 1M2,3-cis/exo-pinanediol.

Whereas 5-norbornene-2,2-dimethanol required formulation in 8.5Mpropylene glycol with 2% 2-pyrrolidone to enable penetration of skin andinduction of melanogenesis (see Example 5 and Tables 8 and 18),2,3-cis/exo-pinanediol induced pigmentation when formulated in only 50%ethanol (Table 18). Biopsies show that similar to induction ofmelanogenesis by 1,2-cis-cyclopentanediol (FIGS. 1C and 6B) and5-norbornene-2,2-dimethanol (FIGS. 1D and 6C), induction ofmelanogenesis by 2,3-cis/exo-pinanediol FIG. 6D) was characterized byproliferation of melanocytes in the basal layer of the epidermis anddistribution of melanin throughout the epidermis. Biopsies from skintreated with 50% ethanol (ETOH) exhibited no such response (FIG. 6A).

TABLE 18 Pigmentation Ratings¹ of Treated Spots Located Anterior (a) toPosterior (d) on Guinea Pigs a b c d 50% ETOH 0.67 ± 0.12 0.25 ± 0.160.08 ± 0.08 0.04 ± 0.04 8.7 M 1,2-cis- 1.83 ± 0.17* 1.92 ± 0.43* 1.92 ±0.20* 1.58 ± 0.24* CPD² 1 M 5- 1.75 ± 0.43* 1.25 ± 0.21* 0.83 ± 0.15*0.29 ± 0.08* NBene-2,2- DM³ 1 M 2,3- 2.00 ± 0.20* 1.75 ± 0.17* 1.75 ±0.17* 1.04 ± 0.32* cs/ex PD⁴ ¹(X ± SE; n = 6); 0 = no change frombackground; +0.25 = slight darkening, indistinct; +0.5 = slightdarkening; +1 = slight moderate darkening; +2 = moderate, evendarkening; +3 = substantial, even darkening; +4 = profound, evendarkening ²1,2-cis-cyclopentanediol in 20% ethanol (ETOH)³5-norbornene-2,2-dimethanol in 8.5 M propylene glycol, 2% 2-pyrrolidoneand 20% ETOH ⁴2,3-cis/exo-pinanediol ([1R,2R,3S,5R]-[−]-pinanediol) in50% ETOH *P < 0.05; Students T-test; significantly different fromETOH-treated site at same position on anterior-posterior axis

EXAMPLE 14

As a continuance of structure activity studies, a variety of pinanediolderivatives and related monocyclic derivatives were examined formelanogenic activity using the S91 cell line and procedures for analysisof tyrosinase described in Example 6. All of the compounds examinedherein were either bicyclic- or monocyclic-monoterpenes (Table 19). Ingeneral, bicyclic-monoterpenes were more potent inducers ofmelanogenesis than monocyclic-monoterpenes, and within each of thesegroups, diols were more potent than alcohols, while non-hydroxylatedcompounds exhibited little or no activity (Table 19).

Bicyclic Monoterpenes

1R,2R,3S,5R)-(−)-pinanediol was only slightly more potent than(1S,2S,3R,5S)-(+)-pinanediol (Table 19), indicating that melanogenicactivity of 2,3-cis/exo-pinanediol is relatively independent ofenantomeric configuration.

(1R)-(−)-trans-pinane-1,10-diol which contains a 2-hydroxymethyl group,exhibited melanogenic potency almost identical to that of(1R,2R,3S,5R)-(−)-pinanediol (Table 19). These results indicate thatmarkedly different pinanediol structures may possess significantmelanogenic activity. Therefore, all pinanediol compounds, includingmethanol and dimethanol substituted pinanediol derivatives are claimedin this invention.

(1S,2S,5S,)-2-hydroxy-3-pinanone was about half as potent as(1R,2R,3S,5R)-(−)-pinanediol (Table 19), indicating that substitution ofa keto group for a hydroxyl group only partially reduces melanogenicactivity. Given this finding, and the fact that keto groups may readilybe converted to hydroxyl groups by chemical or biological processes, itis contemplated that substitution of a keto group for a hydroxyl groupin any of the compounds in this invention may result in retention ofmelanogenic activity. Therefore, all such keto-substituted compounds areclaimed in this invention.

(−)-Isopinochampheol, an alcohol closely related to(1R,2R,3S,5R)-(−)-pinanediol (also known as[−]-2-hydroxyisopinocampheol), possessed considerably less melanogenicactivity (Table 19). In addition, (−)-isopinochampheol resulted indetachment of cells from culture dishes at concentrations where(1R,2R,3S,5R)-(−)-pinanediol was a highly efficacious inducer ofmelanogenesis, indicating that the alcohol was more toxic than the diol.Similar results were obtained for (S)-cis-verbenol, another closelyrelated bicyclic alcohol, and (1R)-(−)-myrtenol, a pinene derivativewhich contains a methanol substituent group (Table 19). Therefore,results for pinanediol derivatives indicate that alcohols are lesspotent inducers of melanogenesis than diols when tested in S91 cells, inagreement with previous results for norbornane derivatives (Example 6,FIG. 2).

However, as noted previously, although 2-norbornanemethanol exhibitedconsiderably less melanogenic activity than 5-norbornene-2,2-dimethanolin S91 cells (Example 6, FIG. 2), it exhibited nearly equivalentcellular melanogenic activity in normal human epidermal melanocytes(Example 7, Table 12). Therefore, it is contemplated that similarfindings may be incurred by (−)-isopinochampheol, (S)-cis-verbenol,(1R)-(−)-myrtenol, and related alcohols when tested in normal humanepidermal melanocytes. Moreover, all alcohol derivatives of thecompounds of this invention are either shown or contemplated to possessvarious degrees of melanogenic activity, and are therefore claimed inthis invention. Nonsubstituted pinane enantiomers exhibited little or nomelanogenic activity.

A mixture of 2,3-cis/exo- and 2,3-trans-bornanediol was found to exhibitapproximately twice as much melanogenic activity as(1R,2R,3S,5R)-(−)-pinanediol (Table 19). Examination of purifiedstereoisomers indicated 2,3-cis/exo-bornanediol was more than twice aspotent as 2,3-trans-bornanediol (Table 19). Borneol, an alcoholderivative, possessed much less activity (Table 19). Results for thesebornane derivatives combined with those for the norbornane derivativesand pinane derivatives provide evidence that any bicyclic or multicycliccompound may provide a suitable framework for incorporation ofsubstituent groups that induce melanogenesis. Therefore, all suchcompounds are claimed in this invention.

Within the bicyclic compounds that were examined, pinane and bornanederivatives (Table 19) were more potent inducers of melanogenesis thannorbornane derivatives (Example 6, FIG. 2; Example 11, Tables 15 and16). Unlike bicyclic norbornanes which contain no methyl substituents,the bicyclic-monoterpene pinanes and bornanes contain three methylsubstituents. Thus, it is contemplated that a range of substituentsincluding but not limited to methyl groups may increase melanogenicactivity of bicyclic compounds.

Monocyclic Monoterpenes

Cis-p-menthane-3,8-diol and trans-p-menthane-3,8-diol were the mostpotent monocyclic monoterpenes examined (Table 19). However, thesepossessed much less melanogenic activity than any of thebicyclic-monoterpene diols examined (Table 19). Similar to results forthe bicyclic-monoterpenes, the alcohols exhibited only low levels ofmelanogenic activity, and were toxic at the higher concentrations tested(Table 19). Moreover, R-(+)-limonene, a non-hydroxylatedmonocyclic-monterpene exhibited little or no melanogenic activity.

Similar to the monocyclic monoterpene alcohols,trans-p-menthane-2,8-diol exhibited much less melanogenic activity thancis-p-menthane-3,8-diol or trans-p-menthane-3,8-diol. However, unlikethe alcohols, trans-p-menthane-2,8-diol was not toxic at the higherconcentrations tested (Table 19). Thus, based on results for bothbicyclic-monocyclic-monoterpenes, it is expected that diols will bepreferable to alcohols as melanogenic agents, not only because they aremore potent, but also because they appear to be less toxic.

Similar to cyclohexanediol, cis-p-menthane-3,8-diol andtrans-p-menthane-3,8-diol possess six member rings. However,cis-p-menthane-3,8-diol and trans-p-menthane-3,8-diol are markedly morepotent than either monocyclic hexanediol or pentanediol (Example 3 andTable 5). Thus, similar to bicyclic compounds, it is contemplated that arange of substituents including but not limited to methyl groups mayincrease melanogenic activity of monocyclic compounds. Enhancement ofmelanogenic potency of aliphatic diols by incorporation of methylsubstituents has been demonstrated previously (e.g., compare2,3-butanediol and 2,3-dimethyl-2,3-butanediol in Example 3, Table 5).

TABLE 19 Fold Induction of Tyrosinase Relative to Controls 0.5 mM 1 mM2.5 mM 5 mM Bicyclic Monoterpenes (1R,2R,3S,5R)-(−)- 5.1X 17.6X   119X50.6X pinanediol (1S,2S,3R,5S)-(+)-  ND¹ 9.9X 68.4X 39.9X pinanediol(1R)-(−)-trans-pinane ND 14.3X  96.0X 36.8X 1,10-diol(1S,2S,5S,)-2-hydroxy- ND 4.1X 37.1X 94.9X 3-pinanone(−)-isopinocamnpheol 2.3X 3.3X  NA² NA (S)-cis-verbenol 1.0X 3.0X 15.5XNA (1R)-(−)-myrtenol³ 13.6X  17.6X   2.2X NA (1R)-(+)-α-pinane 1.2X 0.8X 0.9X NA (1S)-(−)-α-pinane 1.4X 1.1X  1.4X  1.2X 2,3-cis & trans- 9.9X37.3X  99.7X ND bornanediol 2,3-cis/exo-bornanediol 10.2X  28.5X   101XND 2,3-trans-bornanediol 4.7X 12.8X  22.1X ND borneol 3.7X 2.1X  3.7X NDMonocyclic Monoterpenes cis-p-menthane-3,8-diol 1.6X 3.0X 17.1X 11.6Xtrans-p-menthane-3,8-diol 5.2X 13.2X  21.0X  7.3X sobrerol⁴ 1.8X 2.0X 2.3X  3.5X (−)-a-terpineol⁵ 6.2X 7.1X  4.7X NA (1R,2S,5R)-(−)-menthol⁶2.0X 1.3X NA NA (1S,2R,5S)-(+)-menthol⁷ 0.7X 1.3X NA NA R-(+)-limonene⁸1.2X 1.8X  1.6X  1.4X ¹ND: not done ²NA: not analyzed because cells haddetached from culture dishes ³(1R)-2-pinen-10-ol⁴trans-p-menthene-2,8-diol ⁵(S)-p-menth-1-en-8-ol⁶(1R,2S,5R)-2-isopropyl-5-methylcyclohexanol⁷(1S,2R,5S)-2-isoprooyl-5-methylcyclohexanol ⁸(+)-p-mentha-1,8-diene

1. A composition for increasing the melanin content of mammalianmelanocytes comprising: a) an amount, which is effective to increase themelanin content of mammalian melanocytes, of one or more compoundsselected from the group consisting of: (i) bicyclic-monoterpene diols,(ii) pharmaceutically acceptable salts of (i), and (iii) prodrugs of(i); and b) a suitable carrier.
 2. A method for increasing the melanincontent of mammalian melanocytes comprising administering to saidmelanocytes an amount of the composition of claim 1 which is effectiveto increase the melanin content of said melanocytes.
 3. A compositionfor treating a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising: a) an amount, which is effectiveto treat a skin proliferative disorder or a disorder of keratinizationin a mammal, of one or more compounds selected from the group consistingof: (i) bicyclic-monoterpene diols, (ii) pharmaceutically acceptablesalts of (i), and (iii) prodrugs of (i); and b) a suitable carrier.
 4. Amethod for treating a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising administering to a mammal in needof such treatment an amount of the composition of claim 3 which iseffective to treat said skin proliferative disorder or said disorder ofkeratinization in said mammal.
 5. A composition for preventing a skinproliferative disorder or a disorder of keratinization in a mammalcomprising: a) an amount, which is effective to prevent a skinproliferative disorder or a disorder of keratinization in a mammal, ofone or more compounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 6. A methodfor preventing a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising administering to a mammal in needof such preventive treatment an amount of the composition of claim 5which is effective to precent said skin proliferative disorder or saiddisorder of keratinization in said mammal.
 7. A composition for alteringor restoring pigmentation in mammalian skin, hair, wool or furcomprising: a) an amount, which is effective to alter or restorepigmentation in mammalian skin, hair, wool or fur, of one or morecompounds selected from the group consisting of: (i)bicyclic-monoterpene diols, (ii) pharmaceutically acceptable salts of(i), and (iii) prodrugs of (i); and b) a suitable carrier.
 8. A methodfor altering or restoring pigmentation in mammalian skin, hair, wool orfur comprising administering to a mammal in need of such alteration orrestoration an amount of the composition of claim 7 which is effectiveto alter or restore pigmentation in said mammal's skin, hair, wool orfur.
 9. A composition for treating a disease mediated by perturbationsof the NO/cGMP/PKG pathway in a mammal comprising: a) an amount, whichis effective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) bicyclic-monoterpene diols, (ii)pharmaceutically acceptable salts of (i), and (iii) prodrugs of (i); andb) a suitable carrier; said amount being effective to directly stimulateNO synthesis within cells.
 10. A method for treating a disease mediatedby perturbations of the NO/cGMP/PKG pathway in a mammal comprisingadministering to a mammal in need of such treatment an amount of thecomposition of claim 9 which is effective to treat said disease in saidmammal and to directly stimulate NO synthesis within cells.
 11. Acomposition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising: a) an amount, which iseffective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having thefollowing structure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)—CH₂(OH), —(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or—CH₂OR₁, wherein each n is independently an integer from 0-25; each R₁is independently selected from hydrogen; halogen; an acyl or amino acylgroup containing from one atom to twenty atoms, at least one of which iscarbon, nitrogen, oxygen, or sulfur; or a group containing from one atomto twenty atoms, one of which is carbon, nitrogen, oxygen, or sulfur,and R₂ is a linear, branched or unbranched, cyclic, bicyclic orpolycyclic group containing from one atom to fifty atoms, at least oneof which is carbon, nitrogen, oxygen, or sulfur; (ii) unsaturated C₇ toC₅₀ diols having the above structure; (iii) pharmaceutically acceptablesalts of (i); (iv) prodrugs of (i); (v) pharmaceutically acceptablesalts of (ii); and (vi) prodrugs of (ii); and b) a suitable carrier. 12.The composition of claim 11, wherein the C₇ to C₅₀ diol is selected fromthe group consisting of: (a) 5-norbornene-2,2-dimethanol, (b)norbornane-2,2-dimethanol, (c) 2,3-norbornanediol (exo or endo or cis ortrans), (d) 2,3-cis-exo-norbornanediol, (e)2-(propyl-1,2-diol)-norbornane, (f) 2,7-norbornanediol, (g)2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k)2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]), (l) (1R)-(−)-trans-pinane-1,10-diol, (m)2,3-cis/exo-bornanediol, (n) 2,3-trans-bornanediol, (o) camphanediol,(p) camphenediol, and (q) 2,3-trans-pinanediol.
 13. A method fortreating a disease mediated by perturbations of the NO/cGMP/PKG pathwayin a mammal comprising administering to a mammal in need of suchtreatment an amount, which is effective to treat said disease in saidmammal, of one or more compounds selected from the group consisting of:(i) saturated C₇ to C₅₀ diols having the following structure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; or (ii) unsaturated C₇ to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii).
 14. The method of claim 13 wherein the C₇ to C₅₀ diol isselected from the group consisting of: (a) 5-norbornene-2,2-dimethanol,(b) norbornane-2,2-dimethanol, (c) 2,3-norbornanediol (exo or endo orcis or trans), (d) 2,3-cis-exo-norbornanediol, (e)2-(propyl-1,2-diol)-norbornane, (f) 2,7-norbornanediol, (g)2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k)2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]), (l) (1R)-(−)-trans-pinane-1,10-diol, (m)2,3-cis/exo-bornanediol, (n) 2,3-trans-bornanediol, (o) camphanediol,(p) camphenediol, and (q) 2,3-trans-pinanediol.
 15. A composition forincreasing the melanin content of mammalian melanocytes comprising: a)an amount, which is effective to increase the melanin content ofmammalian melanocytes, of one or more compounds selected from the groupconsisting of: (i) saturated C₇ to C₅₀ diols having a bornane structure:(ii) unsaturated C₇ to C₅₀ diols having a bornane structure; (iii)pharmaceutically acceptable salts of (i); (iv) prodrugs of (i); (v)pharmaceutically acceptable salts of (ii); and (vi) prodrugs of (ii);and b) a suitable carrier.
 16. A method for increasing the melanincontent of mammalian melanocytes comprising administering to saidmelanocytes an amount of the composition of claim 15 which is effectiveto increase the melanin content of said melanocytes.
 17. A compositionfor treating a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising: a) an amount, which is effectiveto treat a skin proliferative disorder or a disorder of keratinizationin a mammal, of one or more compounds selected from the group consistingof: (i) saturated C₇ to C₅₀ diols having a bornne structure: (ii)unsaturated C₇ to C₅₀ diols having a bornane structure; (iii)pharmaceutically acceptable salts of (i); (iv) prodrugs of (i); (v)pharmaceutically acceptable salts of (ii); and (vi) prodrugs of (ii);and b) a suitable carrier.
 18. A method for treating a skinproliferative disorder or a disorder of keratinization in a mammalcomprising administering to a mammal in need of such treatment an amountof the composition of claim 17 which is effective to treat said skinproliferative disorder or said disorder of keratinization in saidmammal.
 19. A composition for preventing a skin proliferative disorderor a disorder of keratinization in a mammal comprising: a) an amount,which is effective to prevent a skin proliferative disorder or adisorder of keratinization in a mammal, of one or more compoundsselected from the group consisting of: (i) saturated C₇ to C₅₀ diolshaving a bornane structure: (ii) unsaturated C₇ to C₅₀ diols having abornane structure; (iii) pharmaceutically acceptable salts of (i); (iv)prodrugs of (i); (v) pharmaceutically acceptable salts of (ii); and (vi)prodrugs of (ii); and b) a suitable carrier.
 20. A method for preventinga skin proliferative disorder or a disorder of keratinization in amammal comprising administering to a mammal in need of such preventivetreatment an amount of the composition of claim 19 which is effective toprevent said skin proliferative disorder or said disorder ofkeratinization in said mammal.
 21. A composition for altering orrestoring pigmentation in mammalian skin, hair, wool or fur comprising:a) an amount, which is effective to alter or restore pigmentation inmammalian skin, hair, wool or fur, of one or more compounds selectedfrom the group consisting of: (i) saturated C₇ to C₅₀ diols having abornane structure: (ii) unsaturated C₇ to C₅₀ diols having a bornanestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii); and b) a suitable carrier.
 22. A method for altering orrestoring pigmentation in mammalian skin, hair, wool or fur comprisingadministering to a mammal in need of such alteration or restoration anamount of the composition of claim 21 which is effective to alter orrestore pigmentation in said mammal's skin, hair, wool or fur.
 23. Acomposition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising: a) an amount, which iseffective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having a bornanestructure: (ii) unsaturated C₇ to C₅₀ diols having a bornane structure;(iii) pharmaceutically acceptable salts of (i); (iv) prodrugs of (i);(v) pharmaceutically acceptable salts of (ii); and (vi) prodrugs of(ii); and b) a suitable carrier; said amount being effective to directlystimulate NO synthesis within cells.
 24. A method for treating a diseasemediated by perturbations of the NO/cGMP/PKG pathway in a mammalcomprising administering to a mammal in need of such treatment an amountof the composition of claim 23 which is effective to treat said diseasein said mammal and to directly stimulate NO synthesis within cells. 25.A composition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising: a) an amount, which iseffective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having thefollowing structure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; (ii) unsaturated C₇ to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii); and b) a suitable carrier.
 26. The composition of claim 25,wherein the C₇ to C₅₀ diol is selected from the group consisting of: (a)5-norbornene-2,2-dimethanol, (b) norbornane-2,2-dimethanol, (c)2,3-norbornanediol (exo or endo or cis or trans), (d)2,3-cis-exo-norbornanediol, (e) 2-(propyl-1,2-diol)-norbornane, (f)2,7-norbornanediol, (g) 2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k) 2,3-cis/exo-bornanediol, (l)2,3-trans-bornanediol, (m) camphanediol, and (n) camphenediol.
 27. Amethod for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising administering to a mammal inneed of such treatment an amount, which is effective to treat saiddisease in said mammal, of one or more compounds selected from the groupconsisting of: (i) saturated C₇ to C₅₀ diols having the followingstructure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; or (ii) unsaturated C₇ to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii).
 28. The method of claim 27 wherein the C₇ to C₅₀ diol isselected from the group consisting of: (a) 5-norbornene-2,2-dimethanol,(b) norbornane-2,2-dimethanol, (c) 2,3-norbornanediol (exo or endo orcis or trans), (d) 2,3-cis-exo-norbornanediol, (e)2-(propyl-1,2-diol)-norbornane, (f) 2,7-norbornanediol, (g)2-hydroxy-2-norbornanemethanol, (h)1-(exo-2-norbornyl-)-propan-1,2-diol, (i)1-(endo-2-norbornyl-)-propan-1,2-diol, (j)methyl-5-norbornene-2,3-dimethanol, (k) 2,3-cis/exo-bornanediol, (l)2,3-trans-bornanediol, (m) camphanediol, and (n) camphenediol.
 29. Acomposition for increasing the melanin content of mammalian melanocytescomprising: a) an amount, which is effective to increase the melanincontent of mammalian melanocytes, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having a pinanestructure: (ii) unsaturated C₇ to C₅₀ diols having a pinane structure;(iii) pharmaceutically acceptable salts of (i); (iv) prodrugs of (i);(v) pharmaceutically acceptable salts of (ii); and (vi) prodrugs of(ii); and b) a suitable carrier.
 30. A method for increasing the melanincontent of mammalian melanocytes comprising administering to saidmelanocytes an amount of the composition of claim 29 which is effectiveto increase the melanin content of said melanocytes.
 31. A compositionfor treating a skin proliferative disorder or a disorder ofkeratinization in a mammal comprising: a) an amount, which is effectiveto treat a skin proliferative disorder or a disorder of keratinizationin a mammal, of one or more compounds selected from the group consistingof: (i) saturated C₇ to C₅₀ diols having a pinane structure: (ii)unsaturated C₇ to C₅₀ diols having a pinane structure; (iii)pharmaceutically acceptable salts of (i); (iv) prodrugs of (i); (v)pharmaceutically acceptable salts of (ii); and (vi) prodrugs of (ii);and b) a suitable carrier.
 32. A method for treating a skinproliferative disorder or a disorder of keratinization in a mammalcomprising administering to a mammal in need of such treatment an amountof the composition of claim 31 which is effective to treat said skinproliferative disorder or said disorder of keratinization in saidmammal.
 33. A composition for preventing a skin proliferative disorderor a disorder of keratinization in a mammal comprising: a) an amount,which is effective to prevent a skin proliferative disorder or adisorder of keratinization in a mammal, of one or more compoundsselected from the group consisting of: (i) saturated C₇ to C₅₀ diolshaving a pinane structure: (ii) unsaturated C₇ to C₅₀ diols having apinane structure; (iii) pharmaceutically acceptable salts of (i); (iv)prodrugs of (i); (v) pharmaceutically acceptable salts of (ii); and (vi)prodrugs of (ii); and b) a suitable carrier.
 34. A method for preventinga skin proliferative disorder or a disorder of keratinization in amammal comprising administering to a mammal in need of such preventivetreatment an amount of the composition of claim 33 which is effective toprevent said skin proliferative disorder or said disorder ofkeratinization in said mammal.
 35. A composition for altering orrestoring pigmentation in mammalian skin, hair, wool or fur comprising:a) an amount, which is effective to alter or restore pigmentation inmammalian skin, hair, wool or fur, of one or more compounds selectedfrom the group consisting of: (i) saturated C₇ to C₅₀ diols having apinane structure: (ii) unsaturated C₇ to C₅₀ diols having a pinanestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii); and b) a suitable carrier.
 36. A method for altering orrestoring pigmentation in mammalian skin, hair, wool or fur comprisingadministering to a mammal in need of such alteration or restoration anamount of the composition of claim 35 which is effective to alter orrestore pigmentation in said mammal's skin, hair, wool or fur.
 37. Acomposition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising: a) an amount, which iseffective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having a pinanestructure: (ii) unsaturated C₇ to C₅₀ diols having a pinane structure;(iii) pharmaceutically acceptable salts of (i); (iv) prodrugs of (i);(v) pharmaceutically acceptable salts of (ii); and (vi) prodrugs of(ii); and b) a suitable carrier; said amount being effective to directlystimulate NO synthesis within cells.
 38. A method for treating a diseasemediated by perturbations of the NO/cGMP/PKG pathway in a mammalcomprising administering to a mammal in need of such treatment an amountof the composition of claim 37 which is effective to treat said diseasein said mammal and to directly stimulate NO synthesis within cells. 39.A composition for treating a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal comprising: a) an amount, which iseffective to treat a disease mediated by perturbations of theNO/cGMP/PKG pathway in a mammal, of one or more compounds selected fromthe group consisting of: (i) saturated C₇ to C₅₀ diols having thefollowing structure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; (ii) unsaturated C₇ to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii); and b) a suitable carrier.
 40. The composition of claim 39,wherein the C₇ to C₅₀ diol is selected from the group consisting of: (a)2,3-cis/exo-pinanediol ([1R,2R,3S,5R]-[−]-pinanediol and[1S,2S,3R,5S]-[+]-pinanediol]), (b) (1R)-(−)-trans-pinane-1,10-diol, and(c) 2,3-trans-pinanediol.
 41. A method for treating a disease mediatedby perturbations of the NO/cGMP/PKG pathway in a mammal comprisingadministering to a mammal in need of such treatment an amount, which iseffective to treat said disease in said mammal, of one or more compoundsselected from the group consisting of: (i) saturated C₇ to C₅₀ diolshaving the following structure:

wherein each R is independently selected from R₁; R₂; hydroxyl, methyl,hydroxymethyl, —(CH₂)_(n)CH₃, —(CH₂)_(n)OH, —(CH₂)_(n)OR₁,—(CH₂)_(n)—CH(OH)—CHOH, —(CH₂)_(n)—CH(OH)—CH(OH)R₁,—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH₂(OH),—(CH₂)_(n)—CH(OH)—(CH₂)_(n)—CH(OH)R₁ or —CH₂OR₁, wherein each n isindependently an integer from 0-25; each R₁ is independently selectedfrom hydrogen; halogen; an acyl or amino acyl group containing from oneatom to twenty atoms, at least one of which is carbon, nitrogen, oxygen,or sulfur; or a group containing from one atom to twenty atoms, one ofwhich is carbon, nitrogen, oxygen, or sulfur, and R₂ is a linear,branched or unbranched, cyclic, bicyclic or polycyclic group containingfrom one atom to fifty atoms, at least one of which is carbon, nitrogen,oxygen, or sulfur; or (ii) unsaturated C7 to C₅₀ diols having the abovestructure; (iii) pharmaceutically acceptable salts of (i); (iv) prodrugsof (i); (v) pharmaceutically acceptable salts of (ii); and (vi) prodrugsof (ii).
 42. The method of claim 41 wherein the C₇ to C₅₀ diol isselected from the group consisting of: (a) 2,3-cis/exo-pinanediol([1R,2R,3S,5R]-[−]-pinanediol and [1S,2S,3R,5S]-[+]-pinanediol]), (b)(1R)-(−)-trans-pinane-1,10-diol, and (c) 2,3-trans-pinanediol.